Chemically diverse activity-based probes with unexpected inhibitory mechanisms targeting trypsin-like serine proteases

Abstract

Activity-based probes (ABP) are molecules that bind covalently to the active form of an enzyme family, making them an attractive tool for target and biomarker identification and drug discovery. The present study describes the synthesis and biochemical characterization of novel activity-based probes targeting trypsin-like serine proteases. We developed an extensive library of activity-based probes with “clickable” affinity tags and a diaryl phosphonate warhead. A wide diversity was achieved by including natural amino acid analogs as well as basic polar residues as side chains. A detailed enzymatic characterization was performed in a panel of trypsin-like serine proteases. Their inhibitory potencies and kinetic profile were examined, and their IC50 values, mechanism of inhibition, and kinetic constants were determined. The activity-based probes with a benzyl guanidine side chain showed the highest inhibitory effects in the panel. Surprisingly, some of the high-affinity probes presented a reversible inhibitory mechanism. On the other hand, probes with different side chains exhibited the expected irreversible mechanism. For the first time, we demonstrate that not only irreversible probes but also reversible probes can tightly label recombinant proteases and proteases released from human mast cells. Even under denaturing SDS-PAGE conditions, reversible slow-tight-binding probes can label proteases due to the formation of high-affinity complexes and slow dissociation rates. This unexpected finding will transform the view on the required irreversible nature of activity-based probes. The diversity of this library of activity-based probes combined with a detailed enzyme kinetic characterization will advance their applications in proteomic studies and drug discovery.

Editorial: Mapping enzyme activity: from novel diagnostics to target-based therapeutics, how activity-based probes are improving our understanding of biological catalysts

“…Activity-based probes were first described in 1990 and have become notorious in recent years, where many efforts have been carried out to improve our understanding of protein expression and function in biological systems. The field has advanced the initial goals of the technology, which aimed for the development of inhibitors for a particular target enzyme, towards the usage of activity-based probes to visualize and characterize enzyme activity within a complex proteome ( Fang et al, 2021 ; Ramos-Llorca et al, 2022 ).…”

“…towards the usage of activity-based probes to visualize and characterize enzyme activity within a complex proteome (Fang et al, 2021;Ramos-Llorca et al, 2022).…”